Hemoglobin based bilirubin reference material

ABSTRACT

What is described is a single reference material and method of making useful for calibrating or qualifying instruments that are diagnostic spectroscopically for bilirubin, hemoglobin, and hemoglobin fractions, and, optionally, diagnostic for other blood analytes by sensor means.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/432,448, filed on Apr. 29, 2009, now U.S. Pat.No. 8,119,407 issued on Feb. 21, 2012 which claims priority to U.S.provisional patent application Ser. No. 61/049,197, filed Apr. 30, 2008,the entire contents of each are incorporated herein by reference.

FIELD OF INVENTION

This invention relates to blood-based reference materials useful forcalibrating or qualifying instrument systems for total bilirubin, totalhemoglobin and hemoglobin fractions using spectroscopic measurements.Further, the invention may be used in instruments that have, in additionto CO-Oximetry capabilities, sensors such as electrochemical orenzymatic sensors for measuring pH, blood gasses, e.g., PO₂ and PCO₂,and other blood analytes including electrolytes and metabolites.

BACKGROUND OF THE INVENTION

Determination by automated instrumentation of blood, pH, PO₂, PCO₂,electrolytes, metabolites and CO-Oximetry fractions are routine clinicallaboratory analyses. CO-Oximeter instruments typically measure totalhemoglobin concentration (tHb), and hemoglobin fractions e.g.,oxyhemoglobin (O₂Hb), methemoglobin (MetHb), carboxyhemoglobin (COHb),sulfhemoglobin (SHb), and deoxyhemoglobin (HHb). These fractions arereferred to as CO-Ox fractions or hemoglobin fractions or hemoglobinderivatives. Currently available instruments such as IL 682 and GEM®Premier™ 4000 (Instrumentation Laboratory Company, Bedford, Mass.) havethe ability to measure blood pH, gases, electrolytes, metabolites, totalbilirubin, and/or total hemoglobin and hemoglobin fractions in the sameinstrument.

Blood hemoglobin and hemoglobin fractions absorb visible light withinthe wavelength range of 450-700 nm. For example, a normal oxygenatedblood spectrum has two main peak wavelengths at 542 and 578 nm andabsorbance rapidly decreases close to zero at wavelengths greater than610 nm.

Based on the known hemoglobin fraction wavelength regions and the knownrelationship between hemoglobin concentration and absorbance spectrum,CO-Oximeters analyze blood samples by comparing the sample absorbancespectrum to an instrument calibration set consisting of hemoglobinspectra of known concentrations (FIG. 1) of each individual fraction.The data are analyzed by multicomponent analysis to calculate theconcentrations of total hemoglobin and each hemoglobin fraction presentin a patient's blood sample.

Reference materials generally function to validate the performance of adiagnostic instrument, such as a CO-Oximeter. These materials aretypically aqueous or blood-based materials. As used herein, blood-basedmaterials are materials derived from human or animal blood that includesome component of red blood cells, but not referring to plasma or serummaterials without some component of red blood cells. The blood-basedmaterials allow for the direct match of the spectrum of blood. However,there are no blood-based quality control (QC) or reference materialsavailable that include both hemoglobin fractions and bilirubin due tolimited useful shelf-life caused by, for example, instability ofbilirubin and/or bilirubin. Traditionally, bilirubin is validated usingplasma or serum based reference materials that do not includehemoglobin. Therefore, reference materials for bilirubin on whole/lysedblood CO-Oximetry systems are limited in their ability to establishcorrections for hemoglobin interference due to the lack of bilirubinreference materials that include hemoglobin and hemoglobin fractions.

Bilirubin, a principal component of bile pigment in a body fluid such asblood, is produced by the decomposition of heme from hemoglobin in redblood cells (RBCs). Two main fractions of bilirubin are present inblood. One is free or unconjugated bilirubin (indirect) and the other isconjugated (direct) bilirubin. Conjugated bilirubin is chemically boundto glucuronic acid to render water solubility of bilirubin for excretionfrom the body.

Increased serum bilirubin levels, a combination of direct/conjugated andindirect/unconjugated bilirubin, produce the clinical condition known asjaundice. The liver converts bilirubin to conjugated bilirubin so thatit is excreted from the body. In obstructive jaundice and in liverdisease, excretion or metabolism is impaired and an elevated conjugatedbilirubin fraction occurs. This form of jaundice differs from the causeof neonatal jaundice. The liver of newborns is not fully developed atbirth, and newborns often lack the enzymes necessary to convert theunconjugated form to the conjugated form of bilirubin for excretion.Increased indirect bilirubin is an important measurement for newborns.

Traditional clinical laboratory measurements to assay total bilirubin inblood use the Jendrassik-Gróf principle, where all bilirubin speciesreact with diazonated sulfanilic acid, in the presence ofcaffeine-benzoate acting as a promoter, to yield a red azopyrole. Onaddition of alkaline tartrate to serum or plasma, the color turns blue,which has a maximum absorbance at 598 nm. A disadvantage of this priorart method is that it cannot be completed using whole blood. Serum orplasma must be used, which requires separation from cellular bloodcomponents, typically by centrifugation, before measurement. Morerecently, methods have been developed whereby bilirubin is measured viadirect spectrophotometry on whole blood using analytical instruments,for example, the GEM Premier 4000 cartridge-based system(Instrumentation Laboratory Company, Bedford, Mass.) or the IL Synthesis(Instrumentation Laboratory Company, Bedford, Mass.).

The increasing popularity of the use of direct spectrophotometry inclinical laboratories can be attributed to ease of use, speed ofresults, and convenience. These systems measure whole blood, therebyeliminating the time required to separate blood formed elements fromplasma or serum, using spectrometric measurements to quantify bilirubin,total hemoglobin concentration (tHb), and the hemoglobin fractions O₂Hb,COHb, SHb, and MetHb, i.e., the CO-Ox fractions, and are often coupledto electrochemical sensors to quantify other analytes of interest. Someclinical analyzers only report on a portion of these analytes.CO-Oximeters often lyse RBCs by chemical means, mechanical means, orboth, to reduce scattering effects of cell membranes and to improveoverall accuracy of the measuring system by reducing the backgroundnoise.

Bilirubin, unlike many other blood constituents, is highly unstable inmany reference materials because it is sensitive to light, oxygen, andambient temperatures. For this reason bilirubin based referencematerials typically require refrigerated or frozen storage conditions.In the presence of either light, elevated temperature, or oxygen, theconversion of bilirubin to biliverdin, an analyte which has a verydifferent spectral absorbance than bilirubin, is accelerated. Forexample, in current blood-based reference materials, such as thosedisclosed in U.S. Pat. No. 4,485,174 i.e., blood-based materials thatcontain hemoglobin, oxygen is required in order to provide stable,clinically meaningful levels of oxyhemoglobin, the primary fraction oftotal hemoglobin. The presence of oxygen accelerates the conversion ofbilirubin to biliverdin. To minimize the effect of bilirubin'sinstability, i.e., the conversion of bilirubin to a compound, such asbiliverdin, having a different absorbance, prior art reference materialsfor bilirubin, e.g., those used for CAP NB-surveys, are typicallydispensed in vials and stored frozen. This storage method isunsatisfactory because it increases measurement variability due torigorous handling procedures and provides an opportunity forpre-analytical error. Furthermore, these materials do not provide formeasurement of total hemoglobin and/or hemoglobin fractions togetherwith bilirubin in the same reference material in which the quantities ofthese analytes relate to meaningful clinical levels. Freezing andthawing, as required for many bilirubin reference materials, isunsatisfactory for hemoglobin based reference materials. The risk of aninaccurate measurement is elevated when hemoglobin is measured afterfreezing because the temperature change may induce conversion ofoxyhemoglobin to methemoglobin.

At present, because of the instability of solutions containing bilirubinstored at refrigerated temperatures and the instability of hemoglobinmaterials upon freezing and thawing, a single blood based qualitycontrol or reference material for clinically meaningful concentrationsof bilirubin, total hemoglobin, and its fractions, has not beendeveloped. Organic dye based colored materials are in use by somemanufacturers to simulate the spectral properties of hemoglobin andbilirubin assays. Such dye based products are useful for general qualitycheck of the optical systems but are not very specific in qualifying oridentifying performance quality issues. In addition, dye based qualitymaterials require manufacturer-specific secondary correction factors oranalytical algorithms separate from those used to analyze clinicalsamples to report clinically meaningful hemoglobin or bilirubin results.For this reason, a single blood based reference material, i.e. ablood-based material other than plasma or serum, that providesclinically relevant measurements of both hemoglobin and bilirubinindependent of manufacturer or analytical system has a potential forclinical and commercial interest.

In other words, in spite of the efficiency in qualifying and calibratinginstrument systems that would be provided by such a single referencematerial and the long felt commercial need for such a product, a singleblood based reference material providing clinically relevantconcentrations of bilirubin, hemoglobin and its fractions, withoutrequiring instrument platform-specific secondary correction factors oranalytical algorithms, is not available.

Currently, two separate materials are required for calibrating orrunning quality control on bilirubin, total hemoglobin, and hemoglobinfractions; one for bilirubin and one for total hemoglobin and hemoglobinfractions. Accordingly, the laboratory time required for conductingquality control on bilirubin and hemoglobin is longer than would berequired with a single reference material that includes bilirubin, totalhemoglobin, and hemoglobin fractions. In addition, evaluation ofbilirubin in the presence of clinically relevant concentrations ofhemoglobin provides more accurate corrections for hemoglobininterference on bilirubin, and a more accurate evaluation of ananalyzer's ability to accurately measure these analytes in patientsamples.

SUMMARY OF THE INVENTION

The invention relates to compositions, methods of making, and methods ofusing the composition for quality control of an analytical instrumentfor measurement of bilirubin, total hemoglobin, and hemoglobin fractionsand, optionally, other analytes of interest in body fluids. Theembodiments of the invention have the following features in common, areference material including a blood-based material that haspredetermined quantities of the analytes hemoglobin and/or hemoglobinfractions, and one or more dyes that mimic the absorbance spectra ofbilirubin. The invention is useful as a reference material for each ofthese analytes across a plurality of instrument models from multiplemanufacturers.

In one aspect, the invention relates to a reference material having atleast the following features, (i) at least one component obtained fromblood; and (ii) one or more dyes having an absorbance spectrum similarto bilirubin concentrations at pre-determined clinically relevantlevels. In one embodiment, at least one component obtained from bloodcomprises a pre-determined clinically relevant quantity of totalhemoglobin and hemoglobin fractions.

Alternatively, the reference material according to the inventionincludes at least the following features, (i) at least one componentobtained from blood; (ii) a pre-determined clinically relevant quantityof hemoglobin; and (iii) one or more dyes having an absorbance spectrumsimilar to bilirubin concentrations at pre-determined clinicallyrelevant levels.

The reference material according to the invention includes one or moredyes, for example, chrysophenine, metanil yellow, flavianic acid andcombinations thereof, for example. In one embodiment, the concentrationof chrysophenine is less than about 1000 mg/L, the concentration ofmetanil yellow is less than about 1000 mg/L, and the concentration offlavianic acid is less than about 500 mg/L. The reference materialfurther includes hemoglobin and hemoglobin fractions at pre-determinedlevels that mimic levels that are found in the body fluids of patients.In one embodiment, the hemoglobin fractions are oxyhemoglobin,methemoglobin, deoxyhemoglobin, carboxyhemoglobin, and/orsulfhemoglobin. In one embodiment, according to the invention, the atleast one component obtained from blood is sourced from red blood cells.The reference material is stable at 0-40° C. for at least 2, 6, or 12months or more.

In one embodiment, according to the invention, the reference materialfurther includes a surfactant, such as Brij® 35 (Polyoxyethylene (23)lauryl ether), Brij® 700 (Polyoxyethylene (100) stearyl ether),Arlatone® G (polyoxyethylene fatty glyceride), or Triton® X-100(Polyethylene glycol tert-octylphenyl ether), for example.

In another embodiment, according to the invention, the referencematerial further includes a protein, such as albumin. In a particularembodiment, the concentration of albumin in the reference material is aconcentration that is different than albumin concentrations found in apatient body fluid.

In another aspect, the invention relates to a method for quality controlof an instrument, for example, a clinical analyzer that is capable ofmeasuring bilirubin, total hemoglobin, and hemoglobin fractions. In oneembodiment the method steps include (i) introducing into the analyzer aquality control standard material comprising a pre-determined clinicallyrelevant quantity of hemoglobin, and one or more dyes having anabsorbance spectrum similar to bilirubin concentrations at predeterminedclinical levels, (ii) obtaining an analyzer analysis of the controlstandard material for hemoglobin and/or bilirubin concentrations, and(iii) comparing the analyzer analysis of the hemoglobin concentration orbilirubin concentration with the pre-determined concentrations of thetotal hemoglobin and/or total bilirubin. In one embodiment, the methodfurther includes pre-dissolving the one or more dyes in a surfactantand/or protein solutions, for example in a solution including Brij 35®and albumin. The Brij 35® concentration in the solution may be in therange of about 100-1500 g/L in water, or about 800-1000 g/L in water.The albumin concentration in the solution may be in the range of about1-100 g/L in water, or about 45-55 g/L in water. In one embodiment,pre-dissolving the one or more dyes in a surfactant-containing,protein-containing, or a protein/surfactant-containing solution occursbefore dissolving the one or more dyes in a solution includinghemoglobin. The method according to the invention further requires thatthe one or more of the dyes do not interfere with the analysis of totalhemoglobin or fractions of hemoglobin.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described in more detail below with reference to thefollowing figures, wherein:

FIG. 1 illustrates the spectra of the 4 primary hemoglobin fractions andbilirubin in plasma not in the presence of hemoglobin.

FIG. 2 illustrates the dye spectrum (without hemoglobin) as compared topatient bilirubin in plasma.

FIG. 3 illustrates the correlation between the GEM® Premier™ 4000 vs.ABL 735 analyzers with chrysophenine and metanil yellow dyes evaluatedseparately.

DESCRIPTION OF THE INVENTION

The present invention provides a single blood-based quality controlstandard reference material and method of making and using which, in thepresence of hemoglobin and hemoglobin fractions in blood, mimics thespectrum of bilirubin when the single reference material is used in ananalytical instrument capable of CO-Oximetry.

The problem solved by the invention disclosed herein is making areference material for bilirubin, total hemoglobin, and hemoglobinfractions as a single material such as a single solution that is usefulfor measuring clinically relevant quantities of bilirubin, totalhemoglobin, and hemoglobin fractions in CO-Oximeters on a variety ofinstrument platforms and/or manufactured by more than one company.

As used herein, a clinically relevant reference material may contain acombination of total hemoglobin, hemoglobin fractions, and/or bilirubinin the following concentration ranges (Table I).

TABLE I Target Analyte Ranges for Final Products (HHb = balance) tHbO₂Hb COHb MetHb tBil g dL⁻¹ % % % mg dL⁻¹ Total  0-25  0-100 0-100 0-1000-40 Range Physiological  8-20 60-98 0-20  0-20  0-30 Ranges Normal14-17 94-98 0-3  0-3  0-2  Range (Male) (non- 12-15 smoker) (Female)

The problem solved by the invention relates to a reference material andmethod of making for (i) the analytes bilirubin, total hemoglobin, andhemoglobin fractions, at (ii) clinically meaningful analyteconcentrations that are included in (iii) one reference material, (iv)suitable for serving as a qualifying or calibrating quality controlstandard for bilirubin, total hemoglobin and hemoglobin fractions. Untilthe present invention, clinically relevant quantities could be achievedin standard reference materials for bilirubin, total hemoglobin andhemoglobin fractions only if bilirubin was provided without clinicallyrelevant quantities of hemoglobin in one reference material andhemoglobin and hemoglobin fractions were provided without clinicallyrelevant quantities of bilirubin in another reference material. A singlereference material that combined dye and blood-based materials to mimicclinically relevant concentrations of bilirubin, total hemoglobin, andhemoglobin fractions was not known.

The present invention solves many problems not solved by prior artquality control standard reference material for bilirubin, totalhemoglobin and hemoglobin fractions, despite the long-felt commercialneed for this material. Among the problems solved by the currentinvention is the identification of compounds with bilirubin-likeabsorbance patterns for spectrophotometric scan based measurement.Additional problems solved by the present invention include:

interference with measurement of tHb and CO-Ox fractions;

solubility limitations;

storage stability limitations;

high manufacturing costs;

incompatibility or inaccuracy on some instrument platforms due to theinability to match the spectrum of the bilirubin and the magnitude ofthe bilirubin signal in a background of hemoglobin noise;

incompatibility with some sensors, by inducing excessive signal drift,for example; and

the necessity for multiple reference materials to provide clinicallyrelevant concentrations of total hemoglobin, hemoglobin fractions, andbilirubin.

Moreover, the present invention also solves the problem of providing astable bilirubin absorbance while trying to formulate in a predictablemanner a reference material in the presence of clinically relevantquantities of hemoglobin for analytical systems for the detection ofblood analytes that utilize spectrophotometric analysis, optionally, incombination with electrochemical sensors.

The present invention resolves the long-felt commercial need for stable,blood based quality control standards for bilirubin, total hemoglobinand hemoglobin fractions all in one reference material with acommercially practical shelf life that provides pre-determinedclinically meaningful concentrations of these analytes for qualitycontrol reference materials. The reference material according to theinvention may be used for calibrating clinical analyzers, as acalibration or verification product, or as CAP (College of AmericanPathologists) instrument qualifying survey materials. The stablereference material according to the invention reduces the testing timeand manufacturing costs by combining a bilirubin substitute with otheranalytes e.g., hemoglobin and CO-Ox fractions in a single referencematerial. Bilirubin stabilizing compounds used in prior art qualitycontrol standard materials such as ferrocyanide, EDTA, bilirubin analogsand biliverdin, destabilize hemoglobin fractions, provide insufficientstability in the presence of oxygen, and/or interfere with measurementof other CO-Ox fractions. The reference material according to theinvention may also be used as an internal control to verify spectrometerperformance for bilirubin, total hemoglobin, and hemoglobin fractions.

As used herein, stable means change of no more than 4% for bilirubinfrom the initial bilirubin concentration and no more than 3% for totalhemoglobin from the initial total hemoglobin, when stored from 0-40° C.

Reference materials generally function to validate the performance ofdiagnostic instruments, for example, instruments with CO-Oximeters. Theinvention described herein includes formulations that span theconcentration ranges of analytes which CO-Oximeters are typicallydesigned to measure (Table I), which are broader than the rangestypically observed in patient samples.

The present invention provides a quality control standard referencematerial and method of making, for measurements of bilirubin, totalhemoglobin, and hemoglobin fractions. Typically, dye based productsrequire instrument specific secondary correction factors for the systemto generate meaningful clinical results for hemoglobin or bilirubin.According to the invention, one or more dyes are used to mimic thespectra of bilirubin, where standard blood calculations based onmulticomponent analysis that are used to calculate bilirubin in clinicalsamples can be directly applied to predict the bilirubin equivalentvalue of the dye without application of any secondary correctionfactors. According to the invention, due to the spectral similarities ofthe dyes to bilirubin, no special secondary algorithms are needed toconvert the dye measurements to a meaningful bilirubin result (FIG. 2).Moreover, according to the invention, hemoglobin derived from RBCS(blood-based) is utilized in combination with one or more dyes to make asingle reference material for bilirubin, hemoglobin, and hemoglobinfractions, useful for calibrating or qualifying analytical instrumentshaving CO-Oximetry capabilities. Utilization of hemoglobin isadvantageous solving one problem in prior art reference materials byminimizing the known variability between instrument platforms whenmeasuring total hemoglobin and hemoglobin fractions.

According to the invention, the reference material comprises one or moredyes that mimic the absorbance pattern of bilirubin while havingnegligible absorbance at wavelengths greater than 520 nm (FIG. 2).Significant absorbance at wavelengths greater than 520 nm isdisadvantageous because it would interfere with the measurement ofhemoglobin fractions defeating the advantages of the single referencematerial.

Additionally, in one embodiment according to the invention, in order tomake the reference material, one or more dyes are pre-dissolved in asolution containing one or more proteins such as albumin, for example,bovine serum albumin (BSA) or human serum albumin (HSA) at, for example,room temperature and, optionally, one or more surfactants. Preliminaryobservations identified solubility limitations of the dyes when addeddirectly to aqueous solutions, hemoglobin solutions, deionized water, orto solutions which only contained a protein, such as albumin, or asurfactant. Surprisingly, the addition of exogenous protein, i.e.,proteins in addition to those native proteins found in patient bodyfluids, e.g., albumin in water, significantly increased the solubilityof the dyes, for example, metanil yellow, flavianic acid, andchrysophenine, in the presence of salts, e.g., sodium, potassium, andchloride salts, commonly occurring in human blood and hemoglobin, whilethe proteins natively present in lysed whole blood did not provide asimilar effect on dye solubility. In order to overcome the slowsolubility profile of the dyes in an albumin solution, the dyes werefirst dissolved in a solution containing a surfactant, such as Brij® 35.Therefore the dye was first dissolved in a surfactant solution beforeadding BSA. This mixture was subsequently added to the solution havinghemoglobin, e.g., a hemoglobin concentrate. Any variation on the orderof addition of surfactant, albumin and hemoglobin led to precipitationof the dye, reduced solubility, or a solubility profile that led to aprocessing time that was not commercially acceptable. The concentrationof Brij 35® in the solution used to dissolve the dye prior to additionto the hemoglobin concentration, for example, is in the range of about150 to 1500 g/L, preferably 750 to 1050 g/L, more preferably 900 g/L toensure the concentration of Brij® 35 remained in excess of the criticalmicelle concentration. The concentration of BSA in this same dyeconcentrate solution, for example, is in the range of about 1 to 100g/L, preferably 45 to 55 g/L, more preferably 50 g/L, or exceedsphysiological concentrations of albumin.

The dyes chosen, according to the invention, mimic the spectrum ofbilirubin in a patient sample and do not require a secondary algorithm,separate from those used to analyze clinical samples, to interpret thespectrum to produce meaningful clinical results. According to theinvention, a pre-determined quantity of hemoglobin obtained, forexample, from lysed red blood cells is added to the formulation of thequality control reference material. The quantity of hemoglobin added issufficient to achieve total hemoglobin and hemoglobin fractions in aclinically relevant concentration range, i.e., physiological andpathological concentrations of hemoglobin and hemoglobin fractions (seeTable I).

One or more dyes that are pre-dissolved in a solution containing anexogenous source of protein, are combined with a pre-determined quantityof hemoglobin to result in an absorption spectrum substantially similarto bilirubin over the wavelength range common to most CO-Oximetrysystems.

According to the invention, a single dye or various combinations of dyesare chosen to best-match the absorbance of bilirubin (tBil) at varioustarget analyte concentration ranges for total bilirubin (tBil) in apatent sample (0-40 mg/dL). For example, various dye combinations andtheir concentrations using, for example, metanil yellow, flavianic acid,and chrysophenine, are illustrated in Table II below. Other dyes and dyecombinations are possible and the invention is not limited to theexamples provided.

TABLE II Comparison of dye combinations to best match tBil of patientplasma samples. Metanil Flavianic Sample tBil Yellow Acid Chrysophenine1 13.0 0.2 0.1 0.1 g L⁻¹ 2 12.0 0.0 0.0 0.3 g L⁻¹ 3 33.8 0.0 0.0 1.0 gL⁻¹ 4 11.3 0.0 0.0 0.3 g L⁻¹ 5 8.5 0.0 0.1 0.2 g L⁻¹ 6 16.3 0.3 0.1 0.0g L⁻¹ 7 25.0 0.0 0.0 0.7 g L⁻¹ 8 34.1 0.6 0.2 0.0 g L⁻¹ 9 17.5 0.0 0.00.4 g L⁻¹ 10 17.1 0.0 0.0 0.5 g L⁻¹ 11 25.6 0.1 0.0 0.6 g L⁻¹ 12 24.40.2 0.0 0.4 g L⁻¹ 13 16.7 0.3 0.1 0.1 g L⁻¹ 14 25.2 0.2 0.0 0.5 g L⁻¹ 1529.4 0.5 0.1 0.2 g L⁻¹ 16 19.9 0.4 0.2 0.0 g L⁻¹ 17 13.0 0.0 0.0 0.3 gL⁻¹

The dye selected, the quantity of each dye utilized, and theconcentration of the dyes may be adjusted to reflect the desired levelof tBil in the reference material as shown in Table II, where a singledye or combinations thereof can mimic the bilirubin spectra. The dye ordyes can be scaled to simulate any bilirubin concentration, inparticular, clinically relevant concentrations of bilirubin (see, e.g.,Table I).

The reference material provides sufficient precision and reproducibilityrequired to effectively evaluate an instruments ability to measure totalbilirubin (tBil) (See Table III below).

TABLE III Precision of Total Bilirubin Reference Solutions across 6 GEMPremier 4000 Instruments Chrysophenine Metanil Yellow tHb O₂Hb COHbMetHb HHb tBil tHb O₂Hb COHb MetHb HHb tBil Inst 1 Mean 15.6 97.2 1.60.4 0.8 18.0 15.6 97.2 1.6 0.5 0.7 18.9 SD 0.02 0.14 0.01 0.13 0.01 0.090.05 0.13 0.05 0.07 0.03 0.16 Inst 2 Mean 15.7 97.2 1.8 0.5 0.5 20.215.7 97.5 1.8 0.3 0.4 20.8 SD 0.03 0.12 0.05 0.14 0.02 0.11 0.02 0.160.06 0.11 0.05 0.02 Inst 3 Mean 15.5 97.2 1.8 0.7 0.3 20.1 15.5 97.4 1.90.6 0.2 20.7 SD 0.02 0.05 0.03 0.05 0.04 0.07 0.01 0.14 0.10 0.17 0.060.05 Inst 4 Mean 15.6 96.3 2.3 0.7 0.7 20.4 15.7 96.3 2.3 0.7 0.6 21.6SD 0.02 0.07 0.03 0.05 0.01 0.25 0.03 0.12 0.04 0.05 0.10 0.08 Inst 5Mean 15.6 96.6 2.2 0.8 0.5 19.4 15.6 96.8 2.3 0.6 0.3 20.3 SD 0.03 0.200.08 0.20 0.02 0.11 0.04 0.22 0.05 0.25 0.02 0.26 Inst 6 Mean 15.6 97.31.8 0.5 0.4 19.7 15.6 97.6 1.9 0.3 0.2 20.3 SD 0.05 0.10 0.02 0.09 0.010.23 0.02 0.22 0.04 0.09 0.10 0.52 Overall Mean, mg dL⁻¹ (μM) 19.6(336)  Overall Mean, mg dL⁻¹ (μM) 20.4 (349)  Average Within Run SD, mgdL⁻¹ (μM) 0.16 (2.7)  Average Within Run SD, mg dL⁻¹ (μM) 0.25 (4.3) Instrument to Instrument SD, mg dL⁻¹ (μM) 0.88 (15.1) Instrument toInstrument SD, mg dL⁻¹ (μM) 0.89 (15.2)

Each of the dyes used showed acceptable reproducibility. Through the useof this material it was found that one instrument (Instrument 1)reported significantly lower than the overall population by 10%(p<<0.01) and 9% (p<<0.01) for Chrysophenine and Metanil Yellow,respectively. Using these same instruments to run actual bilirubinsamples it was found that Instrument 1 also recorded significantly belowthe normal population by 9% (p<<0.01), which supports the use of thesereference solutions as an accurate simulation of instrument response totBil.

In one embodiment according to the invention, the reference materialsmay be prepared as calibration materials or quality control materials tosimultaneously calibrate tBil, total hemoglobin, and hemoglobinfractions by spectrometric means, and for blood gas, electrolytes, andvarious metabolites, for example, by enzymatic and electrochemicalsensors.

Preferred reference material compositions, according to the invention,provide commercially acceptable stability in the range of about sixweeks to three years, six weeks to one year, two months to two years,six months to one year, and two months, for example.

According to the invention, components included in the referencematerial do not interfere with electrochemical sensors of the analyticalinstrument in which the reference material is used. Accordingly,calibration or quality control of a variety of sensors and sensor typesmay be simultaneously conducted with the single reference materialaccording to the invention. In addition to pre-determined quantities ofdyes and hemoglobin, components of the reference material may includebuffers, pre-determined quantities of analytes e.g., salts, glucose,lactate, and various gases, and other standard chemicals used instandard reference materials including preservatives, antimicrobials,and surfactants.

In one embodiment according to the invention, the reference materialincludes pre-determined quantities for pH, PCO₂, PO₂, Na⁺, K⁺, Cl⁻,ionized calcium, glucose, lactate, total hemoglobin, hemoglobinfractions, and bilirubin, all of which are in quantities that areclinically meaningful, i.e., mimic physiological and pathologicalconcentrations of these analytes. Reference materials according to theinvention may be used for verifying the accuracy and reliability ofinstruments having sensors and spectrometers for measuring theseanalytes. A particular exemplification is described in Table IV below:

TABLE IV Generic Formulation for Formulation of Final Product Conc UnitsDeionized Water A L Lysed Human RBCs B L O₂Hb Intermediate C % COHbIntermediate D % Brii 35 900 g L⁻¹ DI water Albumin 50 g L⁻¹ DI waterHEPES (pH buffer) 25 mmol L⁻¹ Sodium HEPES 33 mmol L⁻¹ Sodium Lactate 60mmol L⁻¹ NAD 0.7 mmol L⁻¹ Gentamicin 3.33 mg L⁻¹ Example DyeConcentration to Mimic Bilirubin (when used as individual dyes)Simulated Bilirubin 5 15 40 mg dL⁻¹ Chrysophenine 67 200 540 mg L⁻¹Metanil Yellow 70 260 725 mg L⁻¹

A, B, C, and D are varied to reach the desired tHb (deionized water (DI)and lysed RBC's) and hemoglobin fractions (O₂Hb and COHb intermediates)as described, for example, in U.S. Pat. No. 4,485,174, incorporated byreference herein. The dye concentrations listed are provided as anexample, and can be adjusted to simulate intermediate bilirubinconcentrations in the range of about 0-40 mg/dL (shown in Table IV, forexample), as appropriate.

The compatibility of the formulation across multiple instruments thatmeasure bilirubin, for example, GEM® Premier™ 4000 (InstrumentationLaboratory Company, Bedford, Mass.) or ABL 735 (Radiometer, Denmark) areillustrated in FIG. 3. Accordingly, the reference material according tothe invention meets solubility requirements, mimics pre-determinedconcentrations of bilirubin that are clinically meaningful, may be usedon multiple commercial analyzers, does not interfere with the analysisof tHb and its fractions, includes pre-determined clinically meaningfulquantities of total hemoglobin and hemoglobin fractions, and does notinterfere with sensors such as electrochemical sensors, all of which areprovided in a single reference material that can be manufactured at acommercially acceptable cost. This product is stable for commerciallyacceptable time periods for example, as illustrated by therepresentative data in Table V below.

TABLE V Stability evaluation of dyes in blood based matrix Days tHb O₂HbCOHb HHb MetHb tBil Chrysophenine (as measured at 2-8° C. on a GEMPremier ™ 4000) 24 20.0 0.3 98.5 0.0 1.2 24.4 29 20.0 0.5 98.2 0.0 1.225.0 36 19.6 0.1 98.8 0.0 1.1 23.7 41 19.8 0.4 98.4 0.0 1.2 24.3 51 19.90.7 98.4 0.0 0.9 23.6 62 19.9 0.3 98.8 0.0 0.9 24.1 Metanil Yellow (asmeasured at 2-8° C. on an ABL 735) 0 14.9 96 1.4 0.4 2.4 28.0 64 15.1 951.2 0.2 3.6 28.9

Although the present invention has been described with reference tospecific details, it is not intended that such details should beregarded as limitations upon the scope of the invention, except as andto the extent that they are included in the accompanying claims.

It will be apparent to these skilled in the art that variousmodifications and variations can be made to the above-describedmaterials and methods of making without departing from the scope orspirit of the invention.

1. A reference material comprising: (i) a pre-determined clinicallyrelevant quantity of total hemoglobin, or hemoglobin fractions, orcombinations thereof sourced from hemolysed red blood cells; (ii) one ormore dyes having an absorbance spectrum similar to the absorbancespectrum of pre-determined clinical concentrations of bilirubin, whereinsaid one or more dyes do not interfere with the absorbance of thepre-determined quantity of total hemoglobin or the pre-determinedquantity of hemoglobin fractions; (iii) one or more buffers; and (iv)one or more salts.
 2. The reference material of claim 1 furthercomprising, (v) bin analyte selected from the group consisting ofglucose, lactate and calcium.
 3. The reference material of claim 1wherein said one or more salts is selected from the group consisting ofNa, K⁺ and Cl⁻.
 4. The reference material of claim 1 further comprisinga component selected from the group consisting of a preservative, anantimicrobial and a surfactant.
 5. The reference material of claim 4where the surfactant is selected from the group consisting ofpolyoxyethylene (23) lauryl ether, polyoxyethylene (100) stearyl ether,polyoxyethylene fatty glyceride, and polyoxyethylene glycoltert-octylphenyl ether.
 6. The reference material of claim 1 whereinsaid one or more dyes comprise chrysophenine.
 7. The reference materialof claim 1 wherein said one or more dyes comprise metanil yellow.
 8. Thereference material of claim 1 wherein said one or more dyes compriseflavianic acid.
 9. The reference material of claim 1 wherein said one ormore dyes comprise one or more of chrysophenine, metanil and flavianicacid.
 10. The reference material of claim 9 wherein the concentration ofchrysophenine is less than about 1000 mg/ml.
 11. The reference materialof claim 9 wherein the concentration of metanil yellow is less thanabout 1000 mg/ml.
 12. The reference material of claim 9 wherein theconcentration of flavianic acid is less than about 500 mg/ml.
 13. Thereference material of claim 1 wherein said clinically relevant quantityof total hemoglobin is in the range of 8-20 gdl⁻¹.
 14. The referencematerial of claim 1 wherein said pre-determined clinical concentrationof bilirubin is in the range of 0-30 mg dl⁻¹.
 15. The reference materialof claim 1 wherein said hemoglobin fractions comprises 60-98%oxyhemoglobin.
 16. The reference material of claim 1 wherein saidhemoglobin fractions comprises 0-20% carboxyhemoglobin.
 17. Thereference material of claim 1 wherein said hemoglobin fractionscomprises 0-20% methemoglobin.
 18. The reference material of claim 1wherein a target analyte concentration of total hemoglobin in saidreference material is in the range of about 0-25 mg dl⁻¹.
 19. Thereference material of claim 1 wherein a target analyte concentration oftotal bilirubin in said reference material is in the range of about 0-40mg dl⁻¹.
 20. The reference material of claim 1 wherein a targethemoglobin fraction in said reference material comprises a hemoglobinfraction selected from the group consisting of 0-100% oxyhemoglobin,0-100% carboxyhemoglobin, and 0-100% methemoglobin.